Novel peptide ligands of leukocyte integrins

ABSTRACT

The present invention relates to novel peptides which are useful as antagonists of the β 2  integrins, to pharmaceutical compositions comprising these peptides, to the use of the novel peptides for the manufacture of pharmaceutical compositions for the treatment of inflammatory diseases and human leukemias and for inhibiting leukocyte cell adhesion and migration in general, to a method for therapeutic or prophylactic treatment of inflammatory diseases and human leukemias and to the use of the novel peptides as β 2  integrin antagonists for biochemical isolation and purification procedures in vitro.

FIELD OF THE INVENTION

[0001] The present invention relates to novel peptides which are usefulas antagonists of the β₂ integrins, to pharmaceutical compositionscomprising these peptides, to the use of the novel peptides for themanufacture of pharmaceutical compositions for the treatment ofinflammatory diseases and human leukemias and for inhibiting leukocytecell adhesion and migration in general, to a method for therapeutic orprophylactic treatment of inflammatory diseases and human leukemias andto the use of the novel peptides as β₂ integrin antagonists forbiochemical isolation and purification procedures in vitro.

BACKGROUND OF THE INVENTION

[0002] The migration of leukocytes through the body and the variouslymphoid organs is an essential element of the immune system. Whilecirculating in blood or lymphatic vessels, leukocytes are in a restingand low adhesive state. However, when leukocytes are stimulated bysignals from the immune system such as exposure to an immune complex ora chemokine gradient, their integrin adhesion receptors becomeactivated. The activation of the integrins is essential for the manyfunctions of leukocytes.

[0003] Such functions are, for example, binding to antigen-presentingcells, recirculation through lymph nodes and migration out of thevasculature and through the extracellular matrix to sites ofinflammation. The integrin activation needs to be tightly regulated asinappropriate leukocyte adhesion leads to significant injury of normaltissues.

[0004] Leukocytes express a specific subset of the integrin family, theβ₂ integrins of which four members are currently known. They have acommon β₂ chain (CD 18) but different α subunits (α_(L) or CD11a, α_(M)or CD11b, α_(x) or CD 11c, and α_(D) or CD11d) (Gahmberg et al., 1997).The α subunits contain a conserved 200-residue A or I domain, which isessential for binding of most ligands. The crystal structures of Idomains from the α_(L) and α_(M) subunits indicate the presence of acation binding site called the metal-dependent adhesion site. Amino acidsubstitutions in this site abrogate ligand binding (Huang and Springer,1995; Kamata et al., 1995).

[0005] The major ligands of these integrins, the ICAMs, belong to theimmunoglobulin superfamily, and five ICAMs with slightly differentbinding specificities have been described. The expression of ICAM-1 onendothelial cells is subject to stimulation by inflammatory cytokines,which enhances the β₂ integrin-mediated adhesion of leukocytes onendothelial cells. In addition to the ICAMs, fibrinogen and the iC3bcomplement protein are known ligands of the β₂ integrins, particularlyof α_(M)β₂ (Mac-1).

[0006] Because of the importance of the N integrins for leukocytefunction, antagonists of them are potential anti-inflammatory agents.Antibodies to the β₂ integrins or the ICAMs have a therapeutic effect inanimal models of immunological disorders.

[0007] Agents targeting the β₂ integrins could also be valuable in thedevelopment of therapeutic strategies to human leukemias (Calancette etal., 2000). However, only a few small molecule antagonists of the 62integrins have been described so far (Kalen et al., 1999; Kelly et al.,1999). Lack of such compounds has prevented the detailed examination ofthe role of each member of the β₂ integrin family in leukemiadissemination as well in inflammatory diseases. In particular, it wouldbe desirable to design compounds that distinguish between the inactiveand active state of an integrin. Modeling of such small moleculeinhibitors has been hampered by the large size of the peptide ligandsdeveloped so far. Linear peptides are often without a well-definedstructure when free in solution. Among the few β₂ integrin ligandsdiscovered is the 22-amino acid long peptide known as P1 which wasderived from ICAM-2 (Li et al., 1993). This peptide retains theleukocyte integrin-activating effect that is typical for ICAM-2 (Li etal., 1995; Kotovuori et al, 1999). Complementary-determining regions ofanti-02 integrin antibodies have been another source to obtain ligandpeptides, and one of the isolated peptides, 23 amino acids in length,showed similarity to a sequence present on ICAM-1 (Feng et al., 1998).

[0008] To develop smaller peptide ligand leads to the β₂ interns, theinventors screened random peptide libraries displayed on filamentousphage. The phage display technique has previously yielded selectivepeptide ligands to the integrin species α₅β₁ (Koivunen et al., 1994),α_(v)β₃/β₅(Koivunen et al., 1995) and α_(v)β₆ (Kraft et al., 1999).Phage library screenings have also confirmed the earlier findings thatthe tripeptide sequence RGD is a common recognition sequence of a subsetof integins (Pierschbacher and Ruoslahti, 1984). In addition, apparentcharged analogues of RGD, such as RLD, KGD, and NGR, have beendiscovered. The leukocyte integrins α₄β₁, and α₄β₇ are known to have aspecificity for peptides containing another type of tripeptide sequence,LDV (Komoriya et al., 1991).

SUMMARY OF THE INVENTION

[0009] The present inventors have now found that the leukocyte-specificβ₂ integrins recognize a motif comprising three amino acids. Thetripeptide favored by the α_(M) integrin turned out to be a previouslyunknown adhesion motif LLG. An LLG-motif is present on ICAM-1, the major32 integrin ligand, but also on several matrix proteins including vonWirebrand factor and collagens. The inventors developed a novel β₂integrin antagonist peptide termed LLO-Y, which has a compactdisulfide-restrained structure as determined by NMRE Especially thisbicyclic peptide is a potent inhibitor of leukocyte cell adhesion andmigration, and is a novel lead compound for the development ofanti-inflammatory agents.

[0010] It is therefore an object of the present invention to providenovel peptides comprising the structure LLG or a structural or chemicalanalogue thereof, which structure corresponds to the sequence shown inSEQ ID No. 1 of the sequence listing.

[0011] It is another object of the present invention to provide novelpeptides comprising the structure

[0012] CXC LLGCC

[0013] which corresponds to the sequence shown in SEQ ID No. 2 of thesequence listing and wherein X is any amino acid residue, or astructural or chemical analogue thereof

[0014] A further object of the present invention are peptides comprisingthe structure

[0015] CPCFLLGCC

[0016] which corresponds to the sequence shown in SEQ ID No. 3 of thesequence listing, or a structural or chemical analogue thereof.

[0017] The peptides of SEQ ID No. 3 wherein the peptide is structurallyconstrained by two disulfide bonds are preferred. Especially preferredis the peptide with one disulfide bond between the C1 and C8 cysteines,and a second disulfide bond between the C3 and C9 cysteines.

[0018] The present invention also relates to the use of the novelpeptides as pharmaceuticals. Pharmaceutical compositions comprising thenovel peptides in association with a pharmaceutically acceptable carrierform also an object of the present invention.

[0019] The present invention also includes the use of the novel peptidesfor the manufacture of pharmaceutical compositions for the treatment ofinflamatory diseases and human leukemias, and for inhibiting leukocytecell adhesion and migration in general. A further object of theinvention is a method for the therapeutic or prophylactic treatment ofinflammatory diseases and human leukemias which method comprisesadministering a therapeutically or prophylactically effective amount ofa novel peptide according to the invention to a subject in need of saidtreatment.

[0020] The novel peptides according to the invention can also be used asβ₂ integrin antagonists in biochemical isolation and purificationprocedures of different integrin species and different types ofleukocytic cells in vitro.

[0021] The invention is herein below described in more detail referringto the accompanied drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 shows how the binding of LLG-C4-GST nonapeptide to β₂leukocyte integrin and its I-domain is divalent cation-dependent In FIG.1(A), integrin from a blood cell lysate was immunocaptured on microtiterwells using the α_(M) subunit antibody MEM170 or OKM1 or the α_(x)subunit antibody TS2/4. Purified LLG-C4GST or GST control (2 μg/well)was allowed to bind for 60 min in the absence or presence of EDTA. Afterwashing, the bound GST protein was determined by using anti-GSTantibodies. The results show the means ±SD from triplicate wells. Theexperiment was repeated three times with similar results.

[0023] In FIG. 1(B), LLG-C4GST or GST was incubated for 60 min inmicrotiter wells coated with purified α_(M) subunit I-domain. Theconcentrations of GST proteins were as indicated. After washing, thebound GST was determined with anti-GST antibodies. The results are means±SD from triplicate wells. The results were similar in two otherexperiments.

[0024] In FIG. 1(C), LLGGST (10 μg/ml) was incubated in I-domain-coatedwells in the absence or presence of EDTA (2.5 mM) or the LLG-C4 (1)peptide (100 μM) or the inactive LLGC4 (2) peptide (100 μM). After a 60min incubation, the wells were washed and the binding was determinedwith anti-GST antibodies. The results are the means ±SD from triplicatewells.

[0025]FIG. 2 shows how immobilized LLG-C4 supports β₂ integrin-directedcell adhesion.

[0026] In FIG. 2(A), phorbol ester-activated THP-1 cells were allowed tobind for 60 min to microtiter wells coated with LLG-C4-GST, GST, oralbumin. EDTA was included at a 2.5 mM concentration. The bound cellswere determined by the assay measuring cellular phosphatase activity.The data are the means ±SD from triplicate wells.

[0027] Similar results were obtained in six other experiments.

[0028] In FIG. 2(B), THP-1 cells were mixed with each antibody againstthe β₁, β₂, β₃, α_(L), α_(M), or α_(x) integrin subunit as indicated ata concentration of 50 μml. An aliquot of cells was then transferred towells coated with LLG-C4-GST and incubated for 60 min at 37° C.Following washing the bound cells were determined by the phosphataseassay. The results are the mean % adhesion ±SD of 24 independentexperiments each done in triplicate wells.

[0029] In FIG. 2(C), the C(1-8;3-9) and C(1-9;3-8) peptides were coatedon microtiter wells by incubating overnight at a concentration of 40μg/ml in TBS in the absence or presence of glutaraldehyde. Free bindingsites on plastic were then blocked with 5% BSA. THP-1 cells (10 perwell) were allowed to bind for 60 min at 37° C. After a wash with PBS,the bound cells were determined, and the results show the mean±SD oftriplicate wells. The experiment was repeated twice.

[0030] In FIG. 2(D), the α_(x)β₂ integrin-transfected L cells wereallowed to bind to LLG-C4-GST or GST. The 7E4 antibody and EDTA wereused as competitors. The results mean % adhesion ±SD are representativeof three experiments conducted in triplicate wells. The difference inthe binding to LLG-C4-GST vs. GST is statistically significant(p=0.016).

[0031]FIG. 3 shows comparison of activities of cyclic LLG peptides. InFIG. 3(A), TBP-1 cells were mixed in suspension with the peptidecontaining either the C(1-8;3-9) or C(1-9;3-8) disulfide. The finalconcentrations of the peptides are indicated. Cells were then incubatedfor 60 min at 37° C. in microtiter wells coated with LLG-C4 GST. Afterwashing the wells, the bound cells were quantitated by the phosphataseassay. The results are the mean ±SD from triplicate wells. Similarresults were obtained in two other experiments with triplicate wells.

[0032] In FIG. 3(B), THP-1 cell binding to LLG-C4-GST was examined inthe presence of the C(1-8;3-9), CLLGC or the CAAGC peptide. Thesynthetic peptides were used at the concentrations indicated. Followinga 60 min incubation the wells were washed and the bound cells weredetermined by the phosphatase assay. The data show the means ±SD fromtriplicate wells and were similar in two other experiments.

[0033]FIG. 4 shows inhibition of leukocyte cell adhesion to ICAM-1 byLLGC4 peptide. In FIG. 4(A), Jurkat cells were allowed to attach toimmobilized ICAM-1-Fc in microtiter wells in the absence or presence ofLLG-C4 peptides. Following a 45 min incubation, the unbound cells wereremoved by immersing the microtiter plate upside down on a decantercontaining PBS, and the attached cells were stained with Crystal Violet.The results show % cell adhesion ±SD from two experiments each withtriplicate or quadruplicate wells.

[0034] In FIG. 4(B), T cells were allowed to bind to TNF-α-stimulatedEahy endothelial cell monolayers that were grown on microtiter wells.LLG-C4 or RGD-4C was included at a concentration of 50 μM. After a 45min incubation, the unbound cells were removed by immersing themicrotiter plate in a PBS solution, and the bound T cells weredetermined by the phosphatase assay. The data are the mean ±SD oftriplicate wells.

[0035]FIG. 5 shows how ThP-1 cell adhesion on von Willebrand factor andtype IV collagen is blocked by LLG-C4 peptide. In FIG. 5(A), THP-1 cellbinding to von Wirebrand factor was examined in the presence ofantibodies against the f6 (7E4), α_(v)β₃ (LM609) or α_(IIb)β₃ (P2)integrins. Following a 60 min incubation in von Willebrand factor-coatedwells, the bound cells were determined. The data are the mean ±SD oftriplicate wells. The experiment was repeated three times.

[0036] In FIG. 5(B), TRP-1 cells were allowed to bind to microtiterwells coated with von Willebrand factor, type IV collagen, orfibronectin. The C(1-8;3-9) peptide was included as a competitor in theconcentrations described. The bound cells were determined by thephosphatase assay. The data are the mean ±SD of triplicate wells andwere similar in four other experiments.

[0037]FIG. 6 shows that the synthetic LLGC4 peptide prevents adhesionand migration of T1P-1 cells on fibrinogen substratum In FIG. 6(A),THP—I cells were administered together with the C(1-8;3-9) orC(1-9;3-8), or in the absence of peptides, in 11 microtiter wells coatedwith fibrinogen. Following a 60 min incubation the bound cells weredetermined by the phosphatase assay. The results are the mean ±SD oftriplicate wells and were similar in two other experiments.

[0038] In FIG. 6(B), to activate integrins, THP-1 cells were stimulatedboth with phorbol ester (50 nM) and the RGDA4C and C(1-8;3-9) peptides(each 2.5 μM for 1 h. After washing the cells were allowed to bind tofibrinogen-coated wells in the presence of C(1-8;3-9) or RGDA4C or bothpeptides at the concentrations indicated. After a 30-min incubation thebound cells were determined. The results are the mean ±SD of triplicatewells and were similar in two other experiments. At some data points theSD values are too small to be seen.

[0039] In FIG. 6(C), Transwell filters were coated both on the upper andlower surface with fibrinogen, or left uncoated, and then saturated withBSA. TBP-i cells (5×10⁴ per filter) were plated on the upper surface ofthe filter in 10% serum-containing medium. The concentrations ofC(1-8;3-9) and C(1-9;3-8) were 200 μM. Following a 18 hour-culture, thecells migrated underneath the filter were determined. The cells werefixed, stained, and then counted under a microscope. The results showmeans ±SD of at least three experiments.

[0040] In FIG. 6(D), both sides of the Transwell filters were coatedwith LLG-C4GST, GST, fibrinogen, or BSA. A total of 5×10⁴ TP-1 cells wasadministered per filter in 10% serum-containing medium. Cells werecultured for 18 h, and the number of cells migrated to the lower surfaceof filter was counted microscopically. The results show means ±SD of atleast three experiments.

[0041]FIG. 7 shows comparison of structures of cyclic LLG-C4 peptideconformers by NMR. In FIG. 7(A), families of 40 conformations ofC(1-8;3-9) (left) and C(1-9;3-8) (right) are shown. The heavy atoms ofthe disulfide-closed backbones are superimposed in each family and thenthe two families are translated apart for viewing.

[0042] In FIG. 7(B), stereo views of representative solution structuresC(1-8;3-9) (above) and C(1-9;3-8) (below) are shown For thispresentation the two structures were initially superimposed on the mainchain atoms of F4, L5 and L6, and then translated apart for viewing. Inthe C(1-8;3-9) peptide C1 pairs with C8 above and C3 with C9 below thecyclic structure. Likewise, in the C(1-9;3-8) peptide C1 pairs with C9above and C3 with C8 below the ring.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The novel highly specific peptide antagonists containing theunexpected LLG tripeptide binding motif were developed using phagedisplay. The novel peptides inhibit leukocyte adhesion, and whenimmobilized, they support leukocyte adhesion. The most active antagonistCPCFLLGCC (called LLG-C4) is a bicyclic nonapeptide that is structurallyrestrained by two disulfide bonds and contains a LLG tripeptide bindingmotif favoured by the β₂ integrins. The LLG-C4 peptide specificallyblocked the fb integrin-mediated leukocyte adhesion and inhibitedleukocyte binding to their major ligand ICAM-1. Furthermore, like atypical integrin ligand, the peptide supported cell adhesion whenimmobilized on plastic and bound leukocytic cell lines but not cellslacking β₂ integrins. The effectiveness and leukocyte specificity of thepeptide is explained by its ability to interact with the I-domain, whichis a known active site in the leukocyte integrins. Interestingly, notonly ICAM-1 but a number of other adhesion proteins including vonWillebrand factor and type IV collagen contain the consensus PP/XXLLGsequence identified by phage display. Our studies show that vonWillebrand factor and type IV collagen are potential ligands for theleukocyte β₂ integrins.

[0044] The activity of the most active antagonist, LLG-C4 nonapeptide,was strictly dependent on the correct formation of two disulfidebridges. There was a 20-fold difference in the activities of twobiscyclic conformers that differed only in the arrangement of thedisulfide bridges. The more active peptide had a very compact structuredue to “crossing” arrangement of the disulfide bonds as shown by NMRInterestingly, the leucine side chains protrude from the cyclicstructure like antennas suggesting that they can directly interact withthe integrin. The small glycine residue may adjust a correct distancebetween the two leucine side chains.

[0045] Previous studies have indicated that synthetic peptides spanningthe LLG region of ICAM-1 (Ross et al., 1992) or the corresponding regionof ICAM-2 (Li et al., 1993) support-leukocyte adhesion when the peptidesare immobilized on plastic. In soluble forms, the peptides block bindingof leukocytic cells to ICAM-1 expressed on an endothelial cellmonolayer. The LLG-C4 nonapeptide is significantly smaller than thepreviously described peptide ligands for the β₂ integrins and showedhigh activity though lacking a negatively charged amino acid residuesuch as glutamate. Also the pentapeptide CLLGC inhibited cell adhesionThus, β₂ integrin-targeting ligands can be constructed based on thenon-charged LLG motif. This is in accordance with the crystal structuresand structural models of the first Ig domain of ICAM-1 where the LLGsequence is seen as part of a short 0 strand apparently capable ofdirectly contacting with an integrin I-domain (Casasnovas et al., 1998;Bella et al., 1998).

[0046] Alanine scanning mutagenesis studies of individual amino acidswithin the first Ig domain of ICAM-1 have shown that the LLG region isimportant for the integrin binding of ICAM-1. Mutation of one of theleucine residues decreases ICAM-1 binding activity partially andmutation of the glycine completely (Fisher et al., 1997). Because of theinactivity of the glycine-mutated ICAM-1, it has been suggested that theglycine residue does not play a structural role but rather directlyinteracts with the integrin Mutations of the corresponding valine andglycine amino acids to alanines in ICAM-2 also give proteins withimpaired integrin-binding activity (Casasnovas et al., 1999).

[0047] von Willebrand factor contains two LLG sequences but an abilityof these sequences to interact with integrins has not been reported. vonWillebrand factor is a multifunctional adhesive ligand binding severalproteins and prevents bleeding during vascular injury by mediatingplatelet adhesion to exposed subendothelium. It contains two RGDsequences at least one of which is important in binding the plateletintegrin α_(IIbβ) ₃. According to the inventors' knowledge, there are noknown mutations in the LLG sequences associated with bleeding disordersin von Willebrand's disease. The inventors found strong binding ofphorbol ester-activated leukocytic cells to von Willebrand factor. Thebinding appeared to be predominantly mediated by the LLG motif as it wasinhibited by the β₂ integrin-targeting LLG, peptides and by the β₂integrin blocking antibody 7E4 but not by antibodies to the β₃integrins. It is notable that besides the LLG sequences von Willebrandactor contains I domains, similar to those present in the α subunits ofthe β₂ integrins.

[0048] Thus, it is possible that there are intramolecular orintermolecular interactions between the LLG sequences and adjacentI-domains affecting the folding of the protein. If such interactionsoccur, they could in part explain the inactivity of the plasma form ofvon Willebrand factor. Our results suggest that leukocytes can bind toimmobilized form of von Willebrand factor, such as present in vascularsubendothelium or other surfaces, and these interactions could play arole in the initial phases of inflammation.

[0049] Interestingly, the LLGRPGEA sequence of collagen IV a chain thatwe identified by a homology search shows a sequence similarity to thecollagen-like peptide with a critical GFOGER motif (O=hydroxyproline)which binds to α₂β₁ integrin (Enisley et al, 2000). The glutamateresidue of the peptide is seen to coordinate with a metal ion in acrystal structure of the complex between the collagen-like peptide andthe α₂β₁ integrin I-domain. It is possible that upon binding to anintegrin, the glutamate located C-terminal to the LLG motif plays asimilar metal ion-binding role both in ICAM-1 and collagen IV a chain.

[0050] The LLG-C4 nonapeptide is the shortest and most efficient peptideligand yet developed for the β₂ integrins, and provides a lead structurefor the development of β₂ integrin antagonists and potentialanti-inflammatory agents. Because the β₂ integrins are expressed inblood cells and blood cell precursors in the bone marrow, theseintegrins should be readily accessible for targeting by circulatingligands. However, the β₂ integrins are known to exist in an inactivestate and become activated only after physiologic stimuli such as bychemokines or through a contact with antigen-presenting cells.Therefore, it would be desirable and clinically useful to developcompounds binding preferentially to the cells bearing the activatedintegrins. The inventors found the LLGC4 peptide to exhibit suchproperties as the immobilized peptide strongly bound cells only aftertheir integrins were fillly activated The presence of LLG-like sequencesin von Willebrand factor and collagens suggest a novel function for theproteins in mediating not only platelet but also leukocyte adhesion tothe subendothelial matrix of a damaged blood vessel. Consequently, theinhibition of the binding of inflammatory cells and also leukemia cellsto von Willebrand factor and type IV collagens by the peptides of theinvention could be clinically useful. As ICAM-1 functions as thereceptor for rhinoviruses which cause common colds, ICAM-1 mimickingpeptides could also help the development of novel antiviral compounds(Casasnovas et al., 1998; Bella et al., 1998). The peptides of theinvention could also be useful in the treatment of immune diseases,which include, but are not limited to, diabetes, rheumatoid arthritis,Crohn's disease, psoriasis, multiple sclerosis, and transplantrejection.

[0051] In a method for the therapeutic or prophylactic treatment ofinflammatory diseases and human leukemias the therapeutically orprophylactically effective amount of the peptide of the invention to beadministered to a subject in need of said treatment can vary dependingon the condition, body weight, age, etc. of the subject in need of saidtreatment. For a mouse a suitable effective amount is for example 0.2 to1 mg daily, administered intravenously or intraperitoneally, or about 10mg during one week. In order to have an immediate effect on leukocytes,for bigger animals or human beings an effective amount in a single doseis for example 10 to 100 mg i.v.

[0052] The peptides of the present invention can also be used foridentifying leukocyte cell types expressing 0 integrins and may bevaluable for typing inflammatory cells or diagnosing leukemias. Cellsexpressing an activated form of α_(M)β₂ integrin, for example, can beisolated from blood or tissue by passing cell population through thesurface of a column, and then eluting the cells with EDTA or acompetitor peptide.

[0053] This method may be particularly useful for isolating subsets oflymphocyte population expressing a panel of integrins.

[0054] The peptides of the present invention can also be used to promotethe attachment of β₂ integrin expressing cells to a peptide surface oran artificial peptide matrix. Such an artificial matrix may work like abone marrow or lymphatics and help in producing tissue transplants. Onthe other hand, the peptide in soluble form will prevent host reactionsagainst a tissue transplant as the peptide inhibits β₂ integrin directedmigration of macrophages and accompanying inflammatory reactions.

[0055] The following examples illustrate the invention without, however,limiting it in any way.

EXAMPLE 1 Phage Display

[0056] The α_(M)β₂ integrin was purified by antibody affinitychromatography from buffy coats obtained from Finnish Red Cross BloodTransfusion Service as described previously (Li et al., 1995). Integrindiluted in Tris-buffered saline (TBS)/1 mM MnCl₂ was coated ontomicrotiter wells overnight at 4° C. using one jig per well in the firstbiopanning and 100, 10 and 1 ng in subsequent pannings. The wells wereblocked with 5% BSA in TBS for 1 h at 22° C. and then washed five timeswith TBS. Biopanning was performed using CX₇C and CX₉GC phage peptidelibraries essentially as described (Koivunen et al., 1994). Theconstruction of libraries was modified so that the single-stranded DNAencoding degenerate sequences was converted into a double-stranded formusing 5 cycles of PCR with only the reverse primer and then 11 cycleswith both the reverse and forward primer. A total of 6 μg of thedouble-stranded oligonucleotide was purified using QIAGEN PCRpurification kit and ligated with 42 μg of the Fuse5 phage vector. Thenumber of recombinants in the libraries was more than 10⁹. Phagebinding, elution and subsequent amplification in E. coli were repeatedfive times, and after each panning bacterial colonies were picked up andstored in a 10 μl volume of TBS in microtiter wells at −20° C. Fordirect colony sequencing, one td aliquots of the thawed samples weresubjected to PCR with 10 pmol each of the forward primer 5′TAATACGACTCACTATAGGGCAAGCTGATAAACCGATACAATT 3′ and the reverse primer5′CCCTCATAGTTAGCGTAACGATCT 3′. The PCR conditions were 92° C. for 30 s,60° C. for 30 s, and 72° C. for 60 s, and the cycle number was 35. Oneμl aliquot of the PCR reaction was taken for sequencing using 15 pmol ofeither one of the primers and analyzed on an ABI 310 apparatus (PEApplied Biosystems, Foster City, Calif.).

[0057] After the fifh round of selection, the CX₇C library gave a600-fold enrichment and CX₉GC a 1000-fold enrichment of phage bound tothe integrin in comparison to background. Sequencing of the bound phagerevealed altogether only seven different sequences, indicating selectionof specific peptides by the integrin (Table 1). Four of them containedthe LLG tripeptide motif. The two sequences most strongly enriched wereCPCFLLGCC (LLG-C4) and CWKLGSEEEC, each observed 15 times in therandomly selected clones, and these were the only clones remaining aftersearching for high affinity binders by using low integrin coatingconcentrations. Interestingly, LLG-C4 looks like a consensus bindingsequence as all the peptides showed similarities to it with respect tothe conservation of the proline, leucine, glycine, or cysteine residues.TABLE 1 Seven phage sequences bound to the α_(M)β₂ integrin (Mac-1) andtheir alignment with LLG-containing sequences present in cell adhesionproteins CPCFLLGCC (15) CWKLLGSEEEC (15) CWHKDLLGC (4) CWSMELLGCCPPDLFWYC (4) CPEDLYFFC (3) CPEDLYFFC ICAM-1 CDQPKLLGIETPL vonWillebrand Factor-A2 TVGPGLLGVSTLG von Willebrand Factor-D3GRYIILLGKALSV type I collagen-α2 PGPQGLLGAPGIL type IV collagen-α4PGPPGLLGRPGEA osteopontin VICFCLLGITCAI

[0058] Screening protein data bases indicated that the LLG tripeptidesequence is located on the first Ig domain of ICAM-1 just preceding theGlu-34 residue which is critical for ICAM-1 binding to the α_(L)β₂integrn (Staunton et al., 1990; Stanley and Hogg, 1998). The CWKLLGSEEECpeptide showed the highest similarity, five out six consecutive residuesbeing identical to the human ICAM-1 sequence (Table 1). The LLGtripeptide sequence is also contained in domains A2 and D3 of vonWillebrand factor, in a chains of type I and IV collagen, and inosteopontin. None of these LLG-containing sequences, except that ofICAM-1, has been previously reported to contain potential cellattachment sites. von Willebrand factor (Savage et al., 1996) andosteopontin (Helluin et al., 2000) are protein ligands of theRGD-directed 03 integrins but whether they can additionally bind to thef6 integrins is not known.

[0059] Interestingly, type I collagen has recently been shown to be aligand of the α_(x)β₂ integrin (Gamotel et al., 2000).

EXAMPLE 2 Integrin binding assays

[0060] Preparation of GST and Fc fusion proteins

[0061] The nucleotide sequence coding for LLG-C4 was PCR-amplified fromphage DNA with the primers containing a BamH I (5′AGGCTCGAGGATCCTCGGCCGACGGGGCI 3) and an EcOR I site (5′AGGTCTAGAATTCGCCCCAGCGGCCCC 3). The PCR product was purified on anagarose gel, digested with the two restriction enzymes, and ligated intothe PGEX-21K vector (Amersham Pharmacia Biotech, Uppsala, Sweden).Recombinants expressing LLG-C4-GST were verified by DNA sequencing.LLGC4-GST was produced in E. coli strain BL 21 and purified byglutathione afity chromatography followed by dialysis. ICAM-1-Fc fusionprotein containing the five ICAM-1 Ig domains was produced in CHO cellsand purified by protein A affinity chromatography. Approximately 20 mgof LLG-C4-GST was produced, which was more than 95% pure as analyzed bySDS gel electrophoresis on the PhastSystem apparatus (AmershamPharmacia).

[0062] Monoclonal Antibodies

[0063] Antibodies against the integrin β2 subunit were 7E4, 11D3, 3F9,1D10, and 2E7 as described previously (Nortamo ei al., 1988). The antiα_(L) subunit antibodies were TS2/4 and MEM-83 (Monosan, Netherlands).The antibodies OKM1, OKM10 and MEM-170 were against the anti am subunit,and the antibody 3.9 against the α_(x) subunit (Li et al., 1993; Li etal., 1995). The α_(v)β₃ integrin antibody β₂ was purchased fromImmunotech (Marseille, France), and the α_(v)β₃ integrin antibody LM609and the β₁ subunit antibody 6S6 from Chemicon (Temecu Calif.).

[0064] Integrin Binding Assays

[0065] Integrin was immunocaptured on microtiter wells that were coatedwith the α_(m) subunit antibodies OKM1 or MEM170, the α_(x) subunitantibody TS2/4 or non-specific IgG (Dako, Carpinteria, CA). Theantibodies were coated at a concentration of 10 μg/ml in TBS overnightat 4° C.. After saturation of the wells with 5% BSA, a 200 μl aliquot ofthe buffy coat lysate in 1% octylglucoside/1 mM MnCl₂/TBS was allowed toincubate for 2 h at 4° C.. The wells were then washed five times withthe octylglucoside-containing buffer. LLG GST or control GST wasincubated at a concentration of 10 μg/ml in 25 mM octylglucoside TBS/1mM MnCl₂ for an hour.

[0066] Following washing of the wells, the bound GST was determined withanti-GST antibodies (Amersham Pharmacia), which were labeled with anEu³⁺-chelate according to the instructions of the manufacturer (Wallac,Turku, Finland). The Eu³⁺-fluorescence was measured with a 1230 Arcusfluorometer (Wallac). For examination of the I-domain binding, we usedthe recombinant α_(M) I-domain expressed as a GST fusion protein in E.coli as described (Ueda et al., 1994). The fusion protein was purifiedby affinity chromatography on glutathione-coupled beads, and cleavedwith thrombin to release the recombinant I-domain exhibiting a molecularweight of about 23 kDa The I-domain was coated on microtiter wells at aconcentration of 20 μg/ml, and the binding of LLG-C4-GST and GST wasstudied.

[0067] The LLG-C4-GST fusion protein, but not GST alone, was found tohave potent activity and bound to the α_(M)β₂ integrin in a divalentcation-sensitive manner like a typical integrin ligand. Thecation-chelator EDTA inhibited the binding of LLG-C4 GST to theintegrin, which was immunocaptured on microtiter wells with the amsubunit antibody MEM170 or OKM1 (FIG. 1A). Similar EDTA-inhibitablebinding of LLG-C4GST was detected on the α integrin, which was capturedwith the TS2/4 antibody. LLG-C4-GST binding did not differ from GSTcontrol and was not inhibitable by EDTA, when a nonspecific IgG was usedfor immunocapture (not shown).

[0068] We also studied whether the peptide can directly interact withthe I-domain of α_(M)β₂ integrin, the known ligand-binding site.LLG-C4-GST, examined at the concentrations of 0.01-100 μg/ml, showed aconcentration-dependent binding to isolated I-domain of the am subunit(FIG. 1B). GST at the same concentrations did not bind. The ability ofthe I-domain to bind LLG-C4GST was dependent on the Mn2+ cations addedto the binding medium, and chelating M12+with EDTA blocked the binding(FIG. 1C). To show that the I-domain can also bind the LLG-C4nonapeptide and not only the fusion protein, we synthesized thebiscyclic LLG-C4 peptide. However, we had difficulties to prepare anactive and water-soluble peptide, apparently because mixed disulfideseasily formed during air-oxidation. One LLG-C4 (1) preparation washighly active and blocked the ability of the I-domain to bind theLLG-C4-GST (FIG. 1C). The same peptide was also active in cell cultureexperiments. Another preparation, LLG-C4 (2), was inactive apparentlydue to disadvantageous disulfide bonding and did not inhibit LLG-C4-GSTbinding to the I-domain.

EXAMPLE 3 Cell culture and adherence of cell lines to nonapeptide ligand

[0069] The Jurkat T cell leukemia (ATCC no. TIB-152), U-937 histiocyticlymphoma (no. CRL-1593.2), and K562 erytroleukemia (no. 45507) celllines were maintained in RPNH 1640 medium supplemented with 2 mMglutamine, 10 mM HEPES, 1 mM sodium pyruvate, penicillin (100 U/ml),streptomycin (100 μg/ml) and 10% fetal calf serum (FCS). THP-1 monocyticleukemia cells (ATCC TIB-202) were cultured in the same mediumcontaining in addition 0.05 mM 2-mercaptoethanol. The non-leukocyticcell lines Eahy926, HT1080, KS6717 and SKOV-3 were maintained aspreviously described (Koivunen et al., 1999). T cells were isolated fromblood buffy coats by Ficoll-Hypaque centrifugation followed by passagethrough nylon wool columns (Valmu and Gahmberg, 1995). Wild-type mouseL929 cells and α_(x)β₂ integrin-transfected L-cell line were obtainedfrom Dr. Y. van Kooyk University Hospital, Nijmegen, NL).

[0070] Fibrinogen, fibronectin, von Wrlnebrand factor, type IV collagen,GST fusion proteins, Fc fusion proteins or synthetic peptides werecoated on microtiter wells at a concentration of 2 FIG. in 50 μl TBSunless otherwise indicated. A recombinant von Willebrand factor and acapturing antibody to it kindly provided by Drs. J. J. Sixma and Ph. G.de Groot (University Medical Center Utrecht, the Netherlands) were alsoused. To prepare polymerized peptides, glutaraldehyde was added at afinal-concentration of 0.25%. The wells were saturated with 5% BSA andthen washed five times with PBS. Prior to adhesion assays, cells weretreated with 50 nM 40-Phorbol 12,13-dibutyrate or with 200 μM P1 peptide(Kotovuori et al., 1999) in serum-free medium for 30 min at roomtemperature to activate the integrins. Alternatively, cells werestimulated for 60 min at 37° C. with the phorbol ester (50 nM) and theC(1-8;3-9) and RGD4C peptides each at a 2.5 μM concentration, afterwhich the peptides were removed by washing with PBS/2.5 mM EDTA. Cellswere incubated in the micro-titer wells (100 000 cells per well) for 60min at 37° C. in the absence or presence of competing peptides,antibodies or EDTA. Unbound cells were removed by gently washing withPBS and pressing the plate against paper towels. The bound cells weredetermined by an assay measuring cellular phosphatase activity. Briefly,100 01 of 50 mM sodium acetate buffer, pH 5.0, containing 1% TritonX-100 and 6 mg/ml p-nitrophenyl phosphate was added per well andincubated for 1 h at 37° C. The reaction was stopped with 50 μl of 1 MNaOH The absorbance at 405 nm was read on a microplate reader.Alternatively, the attached cells were stained with Crystal Violetessentially as described (Mould et al., 1995).

[0071] To study T cell binding to an endothelial cell monolayer, Eahy926endothelial cells were plated on microtiter plates at a density of 5×10⁴cells per well and grown for three days. To stimulate the production ofICAM-1, the cells were further grown for 16 h in the presence of TNF-αat a concentration of 10 ng/ml. T cells (1.5×10⁵ per well) were allowedto bind to Eahy926 cells first for 30 min at 4° C., then 15 min at 37°C. The unbound T cells were removed by immersing the microtiter plate upside down in PBS. The bound cells were determined by the phosphataseassay.

[0072] Phorbol ester-activated THP-1 monocytic leukemia cellsefficiently bound to LLG-C4GST but not to GST or peptide-GST controls(CLRSGRGCGST, CPPWWSQCGST) coated on microtiter wells (FIG. 2A). EDTA ata concentration of 2.5 mM completely abolished the binding onLLG-C4-GST. Screening with a panel of anti-integrin antibodies indicatedthat the cell adhesion on LLG-C4GST was completely inhibited by theblocking antibody to the β₂ chain, 7E (FIG. 2B). Antibodies to the β₁(6S6) and P3 integrins (LM609, P2) had no effect Partial inhibitionwas-obtained with the β₂ chain antibodies 11D3 and 3F9. The order of thepotency of the three β₂ antibodies is the same as obtained previously ina granulocyte cell aggregation assay, 7E4 being the most activeinhibitor of cell aggregation followed by 11D3 and 3F9 (Nortamo et al.,1988). The inventors also studied the β₂ chain antibodies 1D10, 2F3 and2E7 that can activate the β₂-integrin mediated cell adhesion. Inaccordance, the three antibodies stimulated THP-1 cell adhesion onLLG-C4GST (data not shown).

[0073] Studies with antibodies against the leukocyte integrin a subunitsshowed that a particularly strong inhibition on adhesion was obtainedwith the α_(x) subunit antibody 3.9.

[0074] The α_(M) subunit antibodies OKM10, MEM170 and 60.1 were weaklyinhibitory, whereas the α_(L)-directed antibodies TS1/22 and TS 2/4 hadhardly any effect. TP-1 cells express α_(x) and α_(M) but only littleα_(L), and the antibody inhibition profile is similar to that obtainedfor the inhibition of P1 peptide-stimulated THP-1 cell binding onfibrinogen (Li et al., 1995). Furthermore, the inventors found that theα_(x) antibody 3.9 and the α_(M) antibody OKM10 had a synergistic effectwhen added together, causing a complete inhibition of the cell adhesion.

EXAMPLE 4 Arrangement of the cysteine bonds on the activity of LLG-C4

[0075] As the four cysteines present in the LLG-C4 can theoreticallypair in different ways, we studied how the arrangement of the cysteinebonds affects the activity. For this purpose, we prepared syntheticpeptides with different disulfide configurations.

[0076] Peptides were synthesized using Fmoc-chemistry on an AppliedBiosystems model 433A (Foster City, Calif.). Disulfides were formed byoxidation in 10 mM ammonium bicarbonate buffer (pH 9) overnight Peptideswere then purified by HPLC on an acetonitrile gradient. Generation ofdisulfides was confirmed by mass spectrometry analysis. The C(1-8;3-9)and C(1-9;3-8) peptides with the guided disulfide bridges werecustom-made by Anaspec (San Jose, Calif.). The ACDCRGDCFCG (RGD-4C)peptide (Koivunen et al., 1995) was obtained from Dr. E. Ruoslahti(Bumham Institute, San Diego, Calif.).

[0077] The most active peptide C(1-8;3-9) was obtained by directingformation of one disulfide bond between the C1 and C8 cysteines, and asecond disulfide bond between the C3 and C9 cysteines. The peptideC(1-9;3-8) had the disulfide bridges between the C1 and C9 cysteines,and between the C3 and C8 cysteines. Cells bound to the C(1-8;3-9)disulfide-containing peptide but failed to bind to the conformer withC(1-9;3-8) disulfides (FIG. 2C). Crosslinking of the C(1-8;3-9) peptidewith glutaraldehyde further enhanced cell binding, apparently due tobetter coating of the multimeric peptide, whereas the same treatment ofthe C(1-9;3-8) peptide gave no cell binding.

[0078] In general, the C(1-8;3-9) peptide specifically supported thebinding of β₂ integrin-expressing cell lines such as β₂integrin-transfected L cells and the leukocytic cell lines THP-1, U937and Jurkat. The binding of α_(x)β₂ -transfected L cells to LLGC GST wasinhibited by EDTA and the β₂ integrin blocking antibody 7E4 (FIG. 2D).

[0079] Non-leukocytic cell lines L929, K562, SKOV-3, KS6717 and Eahy96,which do not express β₂ integrins, showed no binding to the peptide orGGL-C4-GCT, whether the cells were pretreated with phorbol ester or not(data not shown).

EXAMPLE 5 Blocking of β₂ integrin-mediated adhesion of leukocyte celllines by LLG-C4 nonapeptide

[0080] The ability of LLG-containing peptides to block leukocyte bindingto adhesion proteins containing or lacking a LLG tripeptide sequence wasexamined. THP-1 cell adhesion on LLG-C4GST was inhibited by theC(1-8;3-9) peptide with an IC₅₀ of 20 μM (FIG. 3A). The other conformer,C(1-9;3-8), was 20-fold less active than C(1-8;3-9). To study whetherthe LLG tripeptide sequence is sufficient for recognition by the 2integrins, the inventors prepared the rib CLLGC peptide containingcysteines at the ends to induce a disulfide-constrained structure. In acontrol peptide the leucines were replaced by alanines. THP-1 celladhesion experiments using the LLG-C4-GST substratum indicated thatCAAGC was a weak competitor of cell adhesion whereas CLLGC readilyinhibited cell adhesion at concentrations of 1 mM or higher, indicatinga specific recognition of the LLG motif by the β₂ integrins (FIG. 3B).

[0081] The ability of LLG-containing peptides to inhibit the α_(L)β₂integrin-mediated binding of Jurkat cells to ICAM-1-Fc recombinantprotein, which includes the first Ig domain with the LLG sequence, wasalso examined. ICAM-1-Fc was directly coated or captured via protein Aon microtiter wells. In both cases a concentration dependent inhibitionby the C(1-8;3-9) peptide on Jurkat cell adhesion was found, and IC₅₀was about 80 μM (FIG. 4A). The C(1-9;3-8) conformer was several foldless active and had hardly any effect. The C(1-8;3-9) peptide similarlyinhibited the binding of freshly isolated T cells to culturedendothelial cells which were stimulated to express ICAM-1 by treatmentwith TNF-α (FIG. 4B). T cells did not bind to unstimulated endothelialcells. As a control, an RGD-containing peptide RGD-C4 had no effect on Tcell binding to endothelial ICAM-1.

[0082] von Willebrand factor and type IV collagen are novel potential β₂integrin ligands as they contain LLG-like peptide motifs. The inventorsfound that phorbol ester-activated TnP-1 strongly bound to bothproteins. The P2integrin antibody 7E4 blocked the THP-1 cell binding tovon Willebrand factor (FIG. 5A) and was nearly as efficient inhibitor asthe cation-chelator EDTA (data not shown). The β₃ integrin antibodiesLM609 and β₂ were without effect. The C(1-8;3-9) peptide was a potentinhibitor of THP-1 cell binding to von Willebrand factor. The peptideinhibited with an IC₅₀ of about 20 μM (FIG. 5B). In addition, the CILGCbut not the CAAGC peptide inhibited at a 500 μM concentration (data notshown). Similar C(1-8;3-9) peptide-mediated inhibition was observed onJurkat cell binding to von Willebrand factor (not shown). THP-1 adhesionon type IV collagen was partially inhibited by the C(1-8;3-9) peptide.The IC₅₀ was about 200 μM. To further study the specificity of the LLGpeptides, we examined THP-1 adhesion to fibronectin, a known ligand ofseveral P1 and P3 integrins. The C(1-8;3-9) peptide showed nosignificant inhibition of fibronectin binding by THP-1 cells. TheC(1-8;3-9) peptide also had no effect on β₁ and β₃ integin-mediatedbinding of RT1080 fibrosarcoma cells on fibronectin or fibrinogen (datanot shown).

[0083] The C(1-8;3-9) peptide was capable of inhibiting THP-1 adhesionto fibrinogen, which does not contain a LLG sequence (FIG. 6A). The lesspotent C(1-9;3-8) conformer had no effect on fibrinogen binding.According to antibody inhibition experiments, the adherence of phorbolester-stimulated THP-1 cells to fibrinogen is predominantly mediated viathe α_(M)β₂ and α_(x) β₂ integrins as previously reported (Li et al.,1995). C(1-8;3-9) similarly inhibited the β₂ integrin-mediatedfibrinogen binding to U937 monocytoid leukemia cells, which express theα_(M)β₂ and α_(x)β₂ integrins (data not shown). As RGD-dependentintegrins can also mediate cell attachment on fibrinogen, the activityof C(1-8;3-9) to that of RGD4C, a potent ligand of several β₁ and 03integrins, was compared. THP-1 cells with low concentrations of theC(1-8;3-9) and RGD-4C peptides were pre-stimulated to fully activateboth the f6 and RGD-dependent integrins. After the peptidepre-stimulation, RGD4C inhibited THP-1 cell adhesion on fibrinogen moreeffectively than C(1-8;3-9) (FIG. 6B). To study whether C(1-8,3-9) andRGD-4C target different integrins, the peptides were given together tocells. The effects of C(1-8;3-9) and RGDA4C were additive and thepeptide combination blocked cell adhesion efficiently.

EXAMPLE 6 Cell migration assay

[0084] The in vitro migration of THP-1 cells was studied in closelyphysiological conditions containing 10% serum to not interfere withadhesive properties of cells. Both the upper and lower surfaces of 8 μmpore size Transwell filters were coated with fibrinogen, LLG-C4-GST, orGST at a concentration of 40 Wgjml overnight at 4° C. Free binding siteswere blocked by incubation with 5% BSA/TBS. THP-1 cells (5×10⁴ in 100μl) were plated on the upper compartment in 10% FCS-containing medium.The C(1-8;3-9) or C(1-9;3-8) peptide were included at a concentration of200 μM The lower compartment was filled with 750 μl of 10%FCS-containing medium. Following a culture for 18 h at 37° C. thefilters were immersed in methanol for 15 min, in water for 10 s, and in0.1% toluidine blue for 5 min. The filters were then washed 3-5 timeswith water until cell staining was clear. Cells were removed from theupper surface of the filter with a cotton swap, and cells migrated onthe lower surface were counted microscopically. Student's t-test wasused for statistical analysis.

[0085] Cells effectively migrated in the presence of 10% serum. TheC(1-8;3-9) peptide at a concentration of 200 μM completely abolished theability of the cells to traverse the filter and bind to its lowersurface (FIG. 6C)>0.005, n=6). The C(1-9;3-8) conformer was less activethan C(1-8;3-9) and inhibited only partially (p=0.01, n=6). The activitydifference between the C(1-8;3-9) and C(1-9;3-8) peptides wassignificant (p=0.003). Cells did not spontaneously drop to the lowerchamber through 8 μM-size pores of the filter. The majority of cellsremained on the upper chamber after a one-day culture. In a reversestrategy where the filter was coated with LLG-C4, cell migration wasstrongly enhanced. Approximately 10-fold more cells migrated on theLLG-C-GST substratum than on control GST substratum (FIG. 6D). Cellmigration on LLG-C4-GST was also more efficient than on fibronectin orfibrinogen coatings.

[0086] C(1-8;3-9) at the 200 p concentration completely suppressed thecell migration on LLG-C4GST (p=0.0026, n=6) (data not shown).,

EXAMPLE 7 NMR analysis of peptides

[0087] C(1-8;3-9) and C(1-9;3-8) peptides were analyzed by NMRspectroscopy to determine whether there are differences in peptideconformations due to the directed arrangement of the disulfide bonds.For the determination, the C(1-8;3-9) peptide was dissolved in DMSO/H₂O(90/10) and C(1-9;3-8) in H₂O at the concentrations of 1-3 mM. Thedifferent solvents were due to the different solubility properties ofthe two peptides. Two-dimensional spectra, acquired with spectrometersoperating at 600 and 800 Miz ¹H-frequency, allowed us to identity 114nuclear Overhauser enhancements (nOes) for C(1-8;3-9) and 85 forC(1-9;3-8) peptide. Forty structures with no restraint violations above0.2 Å were selected from families of 200 structures generated bysimulated annealing (DYANA program).

[0088] The structure determinations of each nonapeptide resulted inwell-defined backbone conformations. RMSD of the main chain atoms was0.4±0.2 Å for C(1-8;3-9) and 0.3±0.2 Å for C(1-9;3-8) calculated fromensembles of 40 sires. For both peptides all main chain dihedrals (p andv are in the favorable and allowed regions of Ramachandran plot Thereare only few nuclear Overhauser enhancements (nOes) to define the sidechain orientation and therefore the side chain dihedrals of F4, L5 andL6, in particular, are dispersed (FIG. 7A).

[0089] The pairing of the disulphides in the two ways influenced thestructure of the nonapeptide considerably. The “crossing arrangement ofdisulphides” of the C(1-8;3-9) peptide constrain the overall structuretighter than the “parallel arrangement of disulphides” of the C(1-9;3-8)peptide. This is reflected by the larger number of nOes observed for theC(1-8;3-9) peptide (114) than for the C(1-9;3-8) peptide (85). There isno bias towards shorter distance restraints in the C(1-8;3-9) peptidecompared with those of the C(1-9;3-8) peptide. As a result of the twoways of pairing the disulphides there are interresidue nOes foundexclusively in one of the structures, 37 in the C(1-8;3-9) peptide and20 in the C(1-9;3-8) peptide.

[0090] The crossing arrangement of disulphides in the C(1-8;3-9) peptideis topologically more complicated than the parallel bridging in theC(1-9;3-8) peptide. In the short nonapeptide the adjacent disulphideswith large van der Waals radii of sulphur atoms give rise to numeroussteric restraints. The residue β₂ also limits conformational freedomwhereas G7 contributes to it The impact of mere topology on the stericrestraints is apparent from representative structures (FIG. 7B). TheC(1-8;3-9) peptide is more compact than the C(1-9;3-8) peptide.Furthermore, there is a continuous hydrophobic surface patch composed ofaliphatic groups of P2, F4 and L5 in the C(1-8;3-9) peptide. Overall,the disulphide bridges and the F4-L6 strand are buckled in theC(1-8;3-9) peptide whereas in the C(1-9;3-8) peptide they are extended.This likely accounts for the poorer water solubility of the C(1-8,3-9)peptide and may contribute to its higher activity.

[0091] Sequence Listing Free Text

[0092] For Seq. ID No. 2:

[0093] Variable aa, Xaa in position 2 can be any amino acid

[0094] Variable aa, Xaa in position 4 can be any amino acid

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[0126] Valmu, L., and Gabmberg, C. G. (1995). Treatment with ocadaicacid reveals strong threonine phosphorylation of CD18 after activationof CD11/CD18 leukocyte integrins with phorbol esters or CD3 antibodies.J. Immunol. 155, 1175-1183.

1 27 1 3 PRT Unknown Peptide of the claimed invention 1 Leu Leu Gly 1 29 PRT Unknown Peptide of the claimed invention 2 Cys Xaa Cys Xaa Leu LeuGly Cys Cys 1 5 3 9 PRT Unknown LLG-C4 peptide of the claimed invention3 Cys Pro Cys Phe Leu Leu Gly Cys Cys 1 5 4 5 PRT Unknown Syntheticpeptide seen in Fig. 3B 4 Cys Leu Leu Gly Cys 1 5 5 5 PRT UnknownSynthetic peptide seen in Fig. 3B 5 Cys Ala Ala Gly Cys 1 5 6 7 PRTUnknown Consensus sequence of ICAM-1 identified by phage display 6 ProPro Xaa Xaa Leu Leu Gly 1 5 7 8 PRT Unknown Sequence of collagen IValpha chain 7 Leu Leu Gly Arg Pro Gly Glu Ala 1 5 8 6 PRT Unknown Motifwhich binds to alpha2beta1 integrin 8 Gly Phe Xaa Gly Glu Arg 1 5 9 43DNA Unknown Forward primer used in direct colony sequencing 9 taatacgactcactataggg caagctgata aaccgataca att 43 10 23 DNA Unknown Reverse primerused in direct colony sequencing 10 cctcatagtt agcgtaacga tct 23 11 11PRT Unknown Stronly enriched sequence 11 Cys Trp Lys Leu Leu Gly Ser GluGlu Glu Cys 1 5 10 12 9 PRT Unknown Phage sequence bound to thealphaMbeta2 integrin (Mac-1) 12 Cys Trp His Lys Asp Leu Leu Gly Cys 1 513 9 PRT Unknown Phage sequence bound to the alphaMbeta2 integrin(Mac-1) 13 Cys Trp Ser Met Glu Leu Leu Gly Cys 1 5 14 9 PRT UnknownPhage sequence bound to the alphaMbeta2 integrin (Mac-1) 14 Cys Pro ProAsp Leu Phe Trp Tyr Cys 1 5 15 9 PRT Unknown Phage sequence bound to thealphaMbeta2 integrin (Mac-1) 15 Cys Pro Glu Asp Leu Tyr Phe Phe Cys 1 516 9 PRT Unknown Phage sequence bound to the alphaMbeta2 integrin(Mac-1) 16 Cys Pro Glu Asp Phe Ile Phe Phe Cys 1 5 17 13 PRT UnknownICAM-1 17 Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu 1 5 10 1813 PRT Unknown von Willebrand Factor-A2 18 Thr Val Gly Pro Gly Leu LeuGly Val Ser Thr Leu Gly 1 5 10 19 13 PRT Unknown von WillebrandFactor-D3 19 Gly Arg Tyr Ile Ile Leu Leu Gly Lys Ala Leu Ser Val 1 5 1020 13 PRT Unknown Type I collagen-alpha2 20 Pro Gly Pro Gln Gly Leu LeuGly Ala Pro Gly Ile Leu 1 5 10 21 13 PRT Unknown Type IV collagen-alpha421 Pro Gly Pro Pro Gly Leu Leu Gly Arg Pro Gly Glu Ala 1 5 10 22 13 PRTUnknown Osteopontin 22 Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys AlaIle 1 5 10 23 29 DNA Unknown Primer containing a BamH I site 23aggctcgagg atcctcggcc gacggggct 29 24 27 DNA Unknown Primer containing aEcoR I site 24 aggtctagaa ttcgccccag cggcccc 27 25 8 PRT Unknown Peptidebound to GST 25 Cys Leu Arg Ser Gly Arg Gly Cys 1 5 26 8 PRT UnknownPeptide bound to GST 26 Cys Pro Pro Trp Trp Ser Gln Cys 1 5 27 11 PRTUnknown RGD-4C peptide 27 Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly 15 10

1. A peptide comprising the structure LLG (SEQ ID NO:1) or a structuralor chemical analogue thereof.
 2. A peptide comprising the structureCXCXLLGCC (SEQ ID NO: 2) wherein X is any amino acid residue, or astructural or chemical analogue thereof.
 3. A peptide comprising thestructure CPCFLLGCC (SEQ ID NO: 3) or a structural or chemical analoguethereof.
 4. The peptide according to claim 2 or 3, wherein said peptideis structurally constrained by two disulfide bonds.
 5. The peptideaccording to claim 4, wherein said peptide contains one disulfide bondbetween the C1 and C8 cysteines, and a second disulfide bond between theC3 and C9 cysteines.
 6. The peptide according to claim 4, wherein saidpeptide contains one disulfide bond between the C1 and C9 cysteines, anda second disulfide bond between the C3 and C8 cysteines.
 7. A peptideaccording to claim 1 for use as a pharmaceutical.
 8. A pharmaceuticalcomposition comprising a peptide according to claim 1 in associationwith a pharmaceutically acceptable carrier.
 9. Use of a peptideaccording to claim 11 for the manufacture of a pharmaceuticalcomposition for the treatment of inflammatory diseases and humanleukemias.
 10. Use of a peptide according to claim 1 as a β₂ integrinantagonist for biochemical isolation and purification procedures ofdifferent integrin species and different types of leukocytic cells invitro.
 11. A method for the therapeutic or prophylactic treatment ofinflammatory diseases and human leukemias which comprises administeringa therapeutically or prophylactically effective amount of a peptideaccording to claim 1 to a subject in need of said treatment.
 12. Use ofa peptide comprising the tripeptide motif LLG or its structural orchemical analogue for the manufacture of pharmaceutical compositions forinhibiting leukocyte cell adhesion and migration.